Project Summary/Abstract The goal of this project is to test the hypothesis that an accumulation of immature autophagosomal membranes induces the non-canonical activation of caspase-8 to switch cytoprotective autophagy to apoptosis for a novel anti-cancer strategy. Autophagy is a double-edged sword in cancer as it can either suppress oncogenesis or promote cancer cell survival. The lack of selective inhibitors of autophagic flux has made it difficult to determine if inhibition of autophagy is a valid cancer strategy. We, and others, have demonstrated that autophagosomal membranes can serve as platforms for an intracellular death-inducing signaling complex (iDISC) that activates caspase-8 independent of death receptor signaling. Mechanistically, the iDISC recruits pro-caspase-8 to autophagosomal membranes by two arms: 1) ATG12-ATG5: FADD: caspase-8; and 2) LC3-II: p62: caspase-8. As ATG12-ATG5 dissociates from the phagophore upon membrane closure and sealed autophagosomes traffic to lysosomes for degradation, we hypothesize that inhibition of phagophore closure will stabilize iDISC assembly for caspase-8 activation. Indeed, our preliminary data reveal that cells deficient in ATG2A/B or VMP1, two regulators of phagophore closure, accumulate immature phagophores that promote iDISC-mediated caspase-8 activation. We propose that elucidation of the molecular mechanisms of phagophore closure will lead to more selective targets for autophagy inhibition and present novel opportunities for cancer therapy. We will investigate our hypothesis in the following specific aims: 1) to demonstrate that the accumulation of immature phagophores initiates iDISC-mediated caspase-8 activation and characterize molecular regulators of non-canonical caspase-8 activation; 2) to test the hypothesis that ATG2A/B and VMP1 regulate phagophore closure through the delivery of ATG9-containing membranes; 3) to demonstrate that impaired phagophore closure can switch autophagy to iDISC-mediated apoptosis in vivo for the suppression of pediatric acute myeloid leukemia (AML) with MLL (mixed lineage leukemia) gene rearrangements.